Bone cells are capable of sensing and responding to mechanical stimuli, but mechanosensitivity begins to decay soon after the stimulus is initiated. Under continued stimulation, a mechanosensory saturated state is reached. As an extension of in vivo work showing that approximately 8 hours of recovery are required to restore mechanosensitivity to bone cells, the long term objective of this project is to determine the cellular and molecular mechanisms involved in bone cell mechanosensory desensitization and subsequent resensitization. They propose that the architecture of the actin cytoskeleton plays an important role in the loss and recovery of mechanosensitivity. The hypothesis is that the shear induced cytoskeletal reorganization process which occurs in response to mechanical stimulation results in a rigid cell that is ineffective in transducing subsequent mechanical signals. Further, stress fiber bundles must be allowed time to disassemble before another robust response can be generated from the next mechanical stimulus. To test these proposals, two hypotheses are presented: (1) Mechanical stimulation causes bone cells to become less sensitive to further stimulation, and a recovery period (no loading) is required to regain mechanosensitivity; and (2) mechanically induced cytoskeletal reorganization into stress fiber bundles causes a decrease in bone cell mechanosensitivity, and disassembly of stress fibers is required to for the cell to respond to subsequent mechanical stimuli. The hypotheses will be tested on cultured osteoblasts and osteocytes using fluid flow induced shear as a mechanical stimulus. Hypothesis 1 will be evaluated by determining (Aim 1) flow duration required for maximal stimulation of mechanotransduction markers: cfos, COX 2, and PGE2; (Aim 2) marker expression during the post flow recovery period; and (Aim 3) marker expression upon reflow at different recovery time points. Hypothesis 2 will be evaluated by determining (Aim 1) the post flow recovery time required for disassembled cytoskeletal architecture; (Aim 2) the association between mechanosensitivity and cytoskeletal architecture; and (Aim 3) changes in mechanosensitivity with agents that either prematurely disassemble or prolong flow induced architecture. Insights into the mechanisms of mechanosensory loss and recovery hold potential in the public health arena for optimizing the positive effects of loading on bone mass, fragility, and fracture risk.